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ISOLATION and CHARACTERISATION of ANTIMYCOBACTERIAL COMPOUNDS from SCHKUHRIA PINNATA (Lam.) Kuntze Ex Thell AGAINST MYCOBACTERIUM SMEGMATIS

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ISOLATION and CHARACTERISATION of ANTIMYCOBACTERIAL COMPOUNDS from SCHKUHRIA PINNATA (Lam.) Kuntze Ex Thell AGAINST MYCOBACTERIUM SMEGMATIS ISOLATION AND CHARACTERISATION OF ANTIMYCOBACTERIAL COMPOUNDS FROM SCHKUHRIA PINNATA (Lam.) Kuntze ex Thell AGAINST MYCOBACTERIUM SMEGMATIS BY MAANO VALERIE MASIPHEPHETHU A DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY IN THE DEPARTMENT OF BIOCHEMISTRY, MICROBIOLOGY AND BIOTECHNOLOGY, SCHOOL OF MOLECULAR AND LIFE SCIENCES, FACULTY OF SCIENCE AND AGRICULTURE, (UNIVERSITY OF LIMPOPO), SOUTH AFRICA. SUPERVISOR: PROF P. MASOKO 2019 Declaration I declare that the dissertation titled: Isolation and characterisation of antimycobacterial compounds from Schkuhria pinnata (Lam.) Kuntze ex Thell against Mycobacterium smegmatis, hereby submitted to the University of Limpopo for the degree Master of Science in Microbiology has not been previously submitted by me to this or any other University and that the work contained herein is my own work in design and execution, and that all materials used are duly acknowledged. Masiphephethu M.V. Date ii Dedication This work is dedicated to my lovely parents (Mr. and Mrs. Masiphephethu), siblings (Fhumulani, Zwivhuya, Martin, Mitambo and Rikonise) and lastly to my daughter (Thalukanyo) and son (Rirhandzu). iii Acknowledgements I would like to offer my sincere gratitude and appreciation to the following: i. It is by his grace, wisdom and strength that the almighty GOD gave to me to go through this study, I thank him and may his blessed name be praised all the times. ii. My gratitude goes to Professor Masoko for allowing me to be under your supervision, your guidance, motivation and patience throughout the study. iii. Dr Z. Mbita and Dr V. Bagla for their assistant with MTT assay. iv. Ms F.D. Mangoakoana for her assistance with anti-inflammatory assay. v. Prof L. McGaw at the University of Pretoria for her assistance in performing cytotoxicity assay. vi. Ms T.G. Ramakadi for assistance with NMR spectroscopy. vii. Prof O. Mazimba at Botswana International University of Science and Technology for assistance with structural elucidation of the compounds. viii. Marumo “my person”, Maryn, Refilwe, Kholofelo, Idris and Nnyadzeni, thank you for assisting me throughout my research. ix. My lovely Family that kept on encouraging, praying and showing pure love, I thank you. x. To the love of my life Rustin Chavani thank you for always believing in me, in times of need you were there giving love, support and comfort. xi. National Research foundation (NRF) for the financial assistance. xii. The University of Limpopo for allowing me to study for my Master’s degree at their facility. iv Table of Contents Declaration ............................................................................................................................ ii Dedication ............................................................................................................................. iii Acknowledgements ............................................................................................................... iv List of figures .................................................................................................................... xiii List of tables ....................................................................................................................... xxiv Abstract................................................................................................................................. 1 Chapter 1 ............................................................................................................................. 3 1. General introduction .......................................................................................................... 3 2. References ....................................................................................................................... 6 Chapter 2 ............................................................................................................................10 2. Literature review ..............................................................................................................10 2.1. Medicinal plants ......................................................................................................... 10 2.2. Phytochemical constituents of medicinal plants ......................................................... 11 2.3. Interaction of plants compounds ................................................................................ 16 2.4. Safety of medicinal plants .......................................................................................... 17 2.5. Bacterial resistance towards antibiotics ..................................................................... 17 2.6. Therapeutic effects of medicinal plants ...................................................................... 18 2.6.1. Antioxidant activity .............................................................................................. 18 2.6.2. Antibacterial activity ............................................................................................ 19 2.6.3. Anti-inflammatory activity .................................................................................... 20 2.7. Methods of studying phytochemicals from medicinal plants .......................................20 2.8. Separation and purification of bioactive compounds .................................................. 23 2.8.1. Thin layer chromatography.................................................................................. 24 2.8.2. High performance liquid chromatography ............................................................ 24 2.8.3. Column chromatography ..................................................................................... 25 2.9. Bioassays for antimicrobial activity ............................................................................ 25 2.9.1. Diffusion methods ............................................................................................... 26 2.9.1.1. Agar disc diffusion assay .................................................................................. 26 2.9.1.2. Agar well diffusion assay .................................................................................. 26 2.9.2. Dilution assays .................................................................................................... 27 2.9.3. Bioautography ..................................................................................................... 28 2.10. Selected plant for this study: Schkuhriav pinnata ....................................................... 29 2.10.1. Description ........................................................................................................ 29 2.10.2. Medicinal uses .................................................................................................. 29 2.11. Microorganism used for the study (Mycobacterium smegmatis) ............................... 30 2.12. Aim and Objectives .................................................................................................. 31 2.12.1. Aim……………………………………………………………………………………...31 2.12.2. Objectives ......................................................................................................... 31 2.13. References .............................................................................................................. 32 Chapter 3 ............................................................................................................................ 47 3. Extraction and Phytochemical screening ....................................................................... 47 3.1. Introduction ............................................................................................................... 47 3.2. Methods and materials .............................................................................................. 48 3.2.1. Plant collection and storage ................................................................................ 48 3.2.2. Extraction procedures ......................................................................................... 49 3.2.3. Phytochemical constituent’s profiles .................................................................... 51 3.2.4. Phytochemical screening using chemical tests .................................................... 51 3.2.5. Determination of total phenolic, flavonoids and tannin content ............................ 53 3.3. Results ...................................................................................................................... 54 3.3.1. Preliminary extraction .......................................................................................... 54 3.3.2. Serial exhaustive extraction ................................................................................ 56 3.3.3. Extraction enrichment procedure......................................................................... 57 3.3.4. Optimal extraction methods ................................................................................. 60 3.3.5. Preliminary serial exhaustive extraction series (1, 2, 3) ....................................... 62 3.3.6. Phytochemical tests ............................................................................................ 65 3.3.7. Determination of total phenolic, flavonoids and tannins content from S. pinnata aqueous acetone extracts. ............................................................................................66 3.4. Discussion ................................................................................................................
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